Genetics

Genome sequences

Grossman lab: Sequencing of the Chlamydomonas genome

The Grossman lab spear-headed the efforts to sequence the 120Mb nuclear genome of the green alga Chlamydomonas reinhardtii. This project has been extremely productive, resulting in numerous papers and websites. A manuscript that describes the full Chlamydomonas genome sequence was published in 2007 (Merchant et al., 2007). This project has also led to the development of the GreenCut, a set of proteins present in green algae and plants but absent from nonphotosynthetic organisms (Grossman et al. 2010); many of these proteins have unknown physiological functions but likely localize in the chloroplasts where they perform important metabolic functions.

Bhaya lab: Sequencing of two Synechococcus strains from hot springs

The Bhaya lab has obtained complete genome sequences of two dominant cyanobacteria (Synechococcus sp.) and an abundant green, non-sulfur photosynthetic bacterium (Roseiflexus sp.) from hot springs in Yellowstone National Park.

Isolation and characterization of mutants

Barton lab: Library of arabidopsis fast neutron mutant lines

The Barton lab is developing a library of thousands of Arabidopsis lines, each carrying a deletion in its genome. The deletions will be generated by fast neutron mutagenesis, which allows more complete coverage of the genome than other mutagenesis strategies. The identity of the deletion in each strain will be determined by a pooling strategy combined with next-generation sequencing.

Evans lab: Library of maize transposon-induced mutants

The Evans lab is generating a collection of insertion mutants in gametophyte-expressed genes of maize using the endogenous maize transposon Activator in maize stocks carrying chromosome arm duplications. The mutant seed stocks will be cross-referenced with mutant phenotype, DNA sequence, and gene expression data.

Jonikas lab: Measuring the growth rates of individual strains grown in a pool.

The Jonikas lab is developing a cutting-edge screening tool for the model green alga Chlamydomonas reinhardtii. This tool will enable simultaneous measurement of growth rates of thousands of individual strains in a pooled culture, through the use of a unique DNA barcode in each strain's genome.

The Grossman lab is screening tens of thousands of Chlamydomonas insertional mutants to isolate strains with mutations in desired genes. A resistance marker is transformed into Chlamydomonas and generates thousands of mutants, each carrying a copy of the resistance marker at a random location in the genome. The mutants are isolated into 96-well plates, and DNA is extracted from pools of 960 mutants. These pools are screened by PCR to identify pools which contain an insertion of the resistance marker in the desired gene. When such pools are found, further PCR allows identification of the precise plates and wells that contain the desired mutants. (Figure 1; Pootakham W et al, Plant Physiol, 2010)

Figure 1. Protocol used to isolate transformants with insertions in specific target genes. Superpools represented ~960 transformants (DNA from transformants grown in 10 microtitre plates, 96 wells each). Primers used were to AphVIII, and the target gene; several different target gene primers are used.